The overall goal of this proposal is to expand the capability for molecular analyses of formalin fixed paraffin-embedded (FFPE) specimens through the use of whole transcriptome profiling with next generation sequencing technology (RNA-Seq). An end-to-end strategy will be developed that includes both experimental and analytical methods to address challenges specific to FFPE samples. This will enable the confident application of RNA-Seq to FFPE material to accurately profile: 1) transcriptional expression, 2) somatic mutations, and 3) chromosomal rearrangements. The strategy for the comprehensive workflow also includes resolving analysis challenges for FFPE RNA- Seq, including developing new bioinformatics tools to enhance the identification of SNV and chromosomal rearrangements. The primary aims of this Phase I application are: Aim 1. Develop a rigorous qualification assay for FFPE RNA that is predictive of sequencing quality. Aim 2. Develop bioinformatics analysis pipeline tailored to FFPE RNA-Seq. Upon the successful completion of these aims, a comprehensive workflow will be established which includes optimized experimental methods and bioinformatics tools to enable use of RNA-Seq for the unbiased discovery of biomarkers. In Phase II, RNA-Seq capabilities will be expanded to further develop and optimize methods, and validate the comprehensive workflow with oncology specimens. This project aligns with Asuragen's expertise and ongoing mission to develop leading-edge bioinformatics tools and to provide high quality NGS assays and NGS products for research and clinical applications.